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浙江大学学报(农业与生命科学版)  2018, Vol. 44 Issue (1): 10-20    DOI: 10.3785/j.issn.1008-9209.2017.05.223
生物科学与技术     
植酸酶基因的定点突变及其在巴斯德毕赤酵母表面展示
余道兵1,程学松2,王群2,史艳可1,张昕1*
1. 浙江农林大学林业与生物技术学院,杭州 311300;2.浙江大学生命科学学院,杭州 310058
Site-directed mutagenesis of Aspergillus niger phytase gene and its cell-surface display on Komagataella phaffii
YU Daobing1, CHENG Xuesong2, WANG Qun2, SHI Yanke1, ZHANG Xin1*
1. College of Forest and Biotechnology, Zhejiang A & F University, Hangzhou 311300, China; 2. College of Life Sciences, Zhejiang University, Hangzhou 310058, China
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摘要:

为解决植酸酶在制粒过程中易变性失活的问题,以黑曲霉(Aspergillus niger)ZJUY中的植酸酶基因phyA为亲本,借助聚合酶链式反应(polymerase chain reaction, PCR)介导的定点突变,对植酸酶基因的关键位点进行密码子优化、引入氢键以及二硫键Cys196-Cys446 的缺失突变;凭借无缝克隆成功将锚定片段FS和携带标签Flag 的目的片段phyA插入酵母表达载体pPICZαC中;采用氯化锂转化法,将构建的8个表达载体pPICZαC-FS/phyA 通过同源重组的方式整合到巴斯德毕赤酵母(Komagataella phaffii)GS115 基因组上。细胞免疫荧光染色和流式细胞仪检测证实,基因改良后的植酸酶在巴斯德毕赤酵母细胞表面成功展示。重组菌株A31、A61和A84的植酸酶活性均可达7 000 U/g。与分泌型植酸酶相比,展示酶具有更好的高温和酸碱耐受性,其中菌株A61在90 ℃水浴处理1 h 后仍有18%的酶活,在pH 1.6~4.0范围内,残余酶活保持在80%以上。菌株A61这一特性可以克服制粒过程中(60~90 ℃)植酸酶变性失活的不足,能够较好地满足动物饲料用酶的需求。

关键词: 植酸酶定点突变表达载体pPICZαC-FS/phyA巴斯德毕赤酵母表面展示    
Abstract:

A full-length sequence of phytase gene (phyA) from Aspergillus niger ZJUY was cloned and modified based on polymerase chain reaction (PCR)-directed mutagenesis, aiming at addressing phytase denaturalization and deactivation during the process of pelleting. The mutation types included codon optimization for key sites, the introduction of hydrogen bond (T295S, Q296R and V43N) and the deletion mutation of disulfide bond (Cys196-Cys446). The anchored-Flo1p (FS) and target-phyA were successfully inserted into the yeast expression vector pPICZαC by seamless cloning. Eight kinds of expression vector pPICZαC- FS/phyA were integrated into the Komagataella phaffii GS115 genome by homologous recombination using lithium chloride transformation method, respectively. Phytases were successfully displayed on the surface of K. phaffii GS115, verified by immunofluorescence staining and flow cytometry. The resulted recombinants A31, A61 and A84 all showed a high phytase activity of 7 000 U/g. In addition, phytase-displaying exhibited better thermostability and pH-stability than those of secretory phytase. Among them, A61 remained 18% phytase activity after water bath at 90 ℃ for 1 h, and still maintained more than 80% of phytase activity at a pH range of 1.6 to 4.0. This characteristic of strain A61 can overcome deactivation during the process of granulating at 60 to 90 ℃ and better meet the demand in animal feed and food processing.

Key words: phytase    site-directed mutagenesis    pPICZαC-FS/phyA    Komagataella phaffii    surface display
收稿日期: 2017-05-22 出版日期: 2017-10-16
CLC:  Q 78  
基金资助: 国家自然科学基金(41271335,31470191,41671314);国家重点基础研究发展计划(973 计划)(2015CB150502)
通讯作者: 张昕(http://orcid.org/0000-0002-8762-171X)     E-mail: zhangxins@126.com
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引用本文:

余道兵, 程学松, 王群, 史艳可, 张昕. 植酸酶基因的定点突变及其在巴斯德毕赤酵母表面展示[J]. 浙江大学学报(农业与生命科学版), 2018, 44(1): 10-20.

YU Daobing, CHENG Xuesong, WANG Qun, SHI Yanke, ZHANG Xin. Site-directed mutagenesis of Aspergillus niger phytase gene and its cell-surface display on Komagataella phaffii. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(1): 10-20.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2017.05.223        http://www.zjujournals.com/agr/CN/Y2018/V44/I1/10

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